AT-hook DNA-binding motif-containing protein one knockdown downregulates EWS-FLI1 transcriptional activity in Ewing’s sarcoma cells

Ewing’s sarcoma is the second most common bone malignancy in children or young adults and is caused by an oncogenic transcription factor by a chromosomal translocation between the EWSR1 gene and the ETS transcription factor family. However, the transcriptional mechanism of EWS-ETS fusion proteins is still unclear. To identify the transcriptional complexes of EWS-ETS fusion transcription factors, we applied a proximal labeling system called BioID in Ewing’s sarcoma cells. We identified AHDC1 as a proximal protein of EWS-ETS fusion proteins. AHDC1 knockdown showed a reduced cell growth and transcriptional activity of EWS-FLI1. AHDC1 knockdown also reduced BRD4 and BRG1 protein levels, both known as interacting proteins of EWS-FLI1. Our results suggest that AHDC1 supports cell growth through EWS-FLI1.


Introduction
Ewing's sarcoma is the second most common bone malignancy in children or young adults. This tumor is caused by a chromosomal translocation of the EWS RNA binding protein 1 (EWSR1) and the E-twenty-six (ETS) transcription factor family, which mainly consists of the Friend Leukemia integration 1 (FLI1), ETS-related gene (ERG), ETS translocation variant 4 (ETV4), or other kinds of ETS transcription factors [1,2]. The EWS-FLI1 fusion protein, U-2 OS cell line were cultured in DMEM with 10% FBS and 1x Penicillin-Streptomycin Solution. Seki, NCR-EW2, and Lenti-X293T cells were spread onto a 0.1% gelatin-coated dish.

Liquid chromatograph-mass spectrometry (LC-MS) analysis and label-free quantification
For BioID analysis, the peptide samples were subjected to a nano-flow reversed-phase (RP) LC-MS/MS system (EASY-nLC™ 1200 System coupled to an Orbitrap Fusion Tribrid Mass Spectrometer: Thermo Fisher Scientific, San Jose, CA) with a nanospray ion source in positive mode. Samples were loaded onto a 75-μm internal diameter × 2-cm length RP C18 precolumn (Thermo Scientific Dionex) and washed with loading solvent before switching the trap column in line with the separation column, a nano-HPLC C18 capillary column (0.075 × 125 mm, 3 mm) (Nikkyo Technos, Tokyo, Japan). A 60-min gradient with solvent B (0.1% Formic acids in 80% acetonitrile) of 5-40% for separation on the RP column equilibrated with solvent A (0.1% formic acid in water) was used at a flow rate of 300 nl/min. MS and MS/MS scan properties were as follows; Orbitrap MS resolution 120,000, MS scan range 350-1500, isolation window m/z 1.6, and MS/MS detection type was ion trap with a rapid scan rate.
All MS/MS spectral data were searched against entries for human in the Swiss-Prot database (v2017-06-07) with a mutant form of E. coli biotin ligase (BirA) using the SEQUEST database search program using Proteome Discoverer 2.2 (PD2.2). The peptide and fragment mass tolerances were set to 10 ppm and 0.6 Da, respectively. For variable peptide modifications, oxidation of methionine and biotinylation of lysine, in addition to carbamidomethylation of cysteine for a fixed modification, were considered. Database search results were filtered by setting the peptide confidence value as high (FDR < 1%) for data-dependent mass analysis data. For label-free quantification, the peptide and protein amount were calculated from the precursor ion intensities using the workflow of Precursor Ions Quantifier in PD2.2. The amount of mutant form of BirA quantified in each analysis was used for the bait normalization, and data from three independent replicas were averaged. ANOVA was performed using the same workflow to calculate the adjusted P values to control experiments (BirA and BirA-Luc2).

Immunoprecipitation
Immunoprecipitation was performed with a slight modification of the following procedure [28]. Cells expressing 3xFLAG-tagged EGFP, ADHC1, or EWS-FLI1 under the control of a Tet-on system which was cultured in a medium containing 1 μg/ml doxycycline for 1 d, were washed by PBS(-) 3 times, collected in PBS(-) after scraping, and centrifuged at 450 g for 10 min at 4˚C. The pellets were treated with 1 ml of hypotonic lysis buffer (10 mM KCl, 10 mM Tris pH 7.5, 1.5 mM MgCl 2 ) supplemented with 1 mM DTT, 1 mM PMSF, 10 μg/ml Leupeptin, and 10 μg/ml Aprotinin for 15 min on ice followed by centrifugation at 400 g for 5 min at 4˚C. Pellets were treated with 500 μl of hypotonic lysis buffer again and mixed by pipetting 10 times, followed by centrifugation at 10000 g for 20 min at 4˚C. Pellets were treated with highsalt extraction buffer (0.42 M KCl, 10 mM Tris pH 7.5, 0.1 mM EDTA, 10% glycerol supplemented with 1 mM PMSF, 10 μg/ml Leupeptin, and 10 μg/ml Aprotinin) with 1 μl Benzonase (70746, Millipore), and gently shaken on an icebox for 30 min, followed by centrifugation at 20000 g for 5 min at 4˚C. Supernatants were diluted by Milli-Q water to adjust to 150 mM salt concentration. 300 μg of nuclear lysate were topped up to 500 μl using IP wash buffer (150 mM KCl, 10 mM Tris pH 7.5, 0.1 mM EDTA, 10% glycerol) supplemented with 1 mM PMSF, 10 μg/ml Leupeptin, and 10 μg/ml Aprotinin. Fifty μl of Anti-FLAG M2 magnetic beads (Cat. No. M8823, Sigma Aldrich) or DDDDK magnetic beads (M185-11, MBL) were washed by PBS(-) once and rotated in 1 ml of 5% BSA/PBS(-) 1 h at 4˚C. The nuclear lysate was mixed and rotated with anti-FLAG magnetic beads for 3 h at 4˚C and washed using 1 ml of IP wash buffer 4 times. Beads were mixed with 50 μl of 2x SDS sample buffer at 95˚C for 5 min. The supernatants were used for Western blotting analysis.
The nuclear lysate was collected using the above method for endogenous protein immunoprecipitation. 500 μg of nuclear lysate was topped up to 500 μl using IP wash buffer and mixed with 10 μg of FLI1 [EPR4646] antibody (ab133485, Abcam) or rabbit normal IgG (Cat.148-09551, Wako pure chemical), followed by rotation at 4˚C for 2 h. The nuclear lysate/IgG was mixed with 25 μl of Pierce™ Protein A/G Magnetic Beads (Cat. 88802, Thermofisher Scientific) with rotation at 4˚C for 2h. The beads were washed with IP wash buffer 4 times and mixed with 50 μl of 2x SDS sample buffer at 95˚C for 5 min. The supernatants were used for Western blotting analysis.

Cell viability assay
Lentiviral-transduced cells were collected without a drug selection, and 1 × 10 3 cells were spread in a 96-well plate. An equal volume of CellTiter-Glo 1 2.0 reagent (Cat. No. G924B, Promega) was transferred into each well and incubated for 5 min. After pipetting each well, the mixture was transferred into a 1.5-ml tube, mixed by a shaker for 10 min at room temperature, and luminescence was measured by a GloMax 1 20/20 Luminometer (Cat. No. E5311, Promega). For measuring measure apoptotic activity, an equal volume of Caspase-Glo 1 3/7 Assay System (Cat. No. G8090, Promega) was transferred into each well and incubated for 1 h, and measured by a GloMax 1 20/20 Luminometer. For the spheroid formation assay, lentiviral-transduced cells were collected, and 1 × 10 4 cells were spread in a PrimeSurface96U (Cat. No. MS-9096U, Sumitomo Bakelite). The medium was changed every 2 d, and photos were taken by an All-in-One fluorescence microscope BZ-810X (Keyence).

Scratch wound healing assay
Lentiviral-transduced cells were cultured in a 12-well plate. The cell layer was scratched by a 1-ml tip, washed with PBS(-) twice, the medium was replaced with DMEM without FBS, and photos were taken by the BZ-810X.

Biotinylation of proximal proteins by BioID in A673 cells
For BioID-tagged EWS-ETS fusion protein expression, we constructed the piggyBac system under the control of the Tet-on system to regulate the gene expression. BioID-tagged EWS-FLI1, EWS-ERG, or EWS-ETV4-expressing plasmids were transfected into A673 cells with a hyperactive piggyBac transposase previously generated for applications in mammalian genetics [22]. After a puromycin selection, cells expressed each BioID-tagged gene by doxycycline with biotin. BioID alone or BioID-tagged Luc2 (firefly luciferase) were used as a negative control and labeled biotin to proximal proteins in all cell fractions (Fig 1A). In addition, BioID-tagged EWS-ETS fusion proteins were mainly localized in the nuclei. Next, we checked whether BioID-tagged EWS-ETS fusion proteins could biotinylate proximal proteins in A673 cells by Western blotting (Fig 1B). Streptavidin-HRP staining confirmed the appearance of various biotinylation bands. BioID-tagged EWS-ETS fusion proteins were highly detected compared to endogenous EWS-FLI1 in a medium containing 1 μg/ml doxycycline (S1A Fig). NKX2-2 and NR0B1 downregulated in cells expressing BioID-tagged EWS-ETS fusion proteins. In addition, BioID-tagged EWS-ETS fusion protein expression reduced cell growth (S1B Fig). We prepared three independent biological replicates for each cell line, collected biotinylated proteins by a streptavidin sepharose set up using the Couzens et al. method [27], and identified proteins by mass spectrometry analysis (Fig 1C and S3 Table). The negative controls were used BioID or BioID-tagged Luc2. For label-free quantification, peptide and protein amount were calculated from precursor ions (Materials and methods) and normalized using BirA(R118G) in each analysis. Each abundance intensity was calculated for each ratio using both BioID and BioID-Luc2. Out of 3879 proteins detected in all samples, 193 proteins were identified as proximal proteins shared from the three fusion proteins (Abundance ratio: each fusion proteins list compared to BioID and BioID-Luc2 > 5, Abundance Ratio Adj. p-value < 0.05). The STRING To characterize between EWS-ETS fusion proteins and an uncharacterized protein, we first focused on AHDC1 protein. AHDC1 was contained in the BioID-tagged EWS-FLI1 and EWS-ERG protein samples (Fig 1C). However, AHDC1 did not significantly differ in the BioID-tagged EWS-ETV4 protein list.
To determine whether AHDC1 is a proximal protein of EWS-ETS fusion proteins, we purified the biotinylated proteins again and detected AHDC1 (S4A Fig and Fig 2A). The intensity of AHDC1 in the EWS-ETS protein sample was higher than in each BioID and BioID-Luc2 sample. Next, immunoprecipitation for AHDC1 was performed using FLAG-tagged AHD-C1-expressing cells (Fig 2B). FLAG-tagged AHDC1 was immuno-precipitated with endogenous EWS-FLI1 protein compared to FLAG-tagged EGFP. This result was reproduced using different magnetic beads (S4B Fig). FLAG-tagged EWS-FLI1 was also immunoprecipitated with endogenous AHDC1 compared to FLAG-tagged EGFP (Fig 2C). Moreover, endogenous EWS-FLI1 immunoprecipitants were included in AHDC1 with BRD4 and BRG1 (Fig 2D).

AHDC1 knockdown affects gene expression of EWS-FLI1 target genes
To evaluate whether AHDC1 affects gene expression of EWS-FLI1, we treated A673 cells with siRNA for the AHDC1 knockdown experiment. AHDC1 knockdown showed reduced EWS--FLI1 protein expression level but not EWSR1 (Fig 3A). The nuclear receptor NR0B1 and the homeobox transcription factor NKX2-2 were up-regulated in Ewing's sarcoma [30][31][32]. NR0B1 and NKX2-2 protein expression levels were reduced in siAHDC1-treated cells. Silencing of EWS-FLI1-bound GGAA microsatellite by a dCas9-KRAB system showed downregulation of NKX2-2 and SOX2 protein expression in A673 and SKNMC cells [33]. However, AHDC1 knockdown did not change the SOX2 protein level in A673 cells. We also tested whether AHDC1 knockdown reduces protein expression levels in other Ewing's sarcoma cell lines. For this purpose, we treated Seki or NCR-EW2 cell lines, both of which have been established as Ewing's sarcoma cells, with siAHDC1 RNA [20,34]. EWS-FLI1 and NR0B1 were downregulated in both cell lines (S4C and S4D Fig). NKX2-2 was only downregulated in NCR-EW2 cells. Ten μg of proteins and one-tenth of pulldown input were used as a whole-cell lysate and a biotinylated protein sample, respectively. Band intensity was compared as a BioID or BioID-tagged Luc2. GAPDH antibody was used as a negative control. (B) Western blotting analysis of co-immunoprecipitated samples. 300 μg of nuclear lysate was mixed with FLAG M2 magnetic beads for immunoprecipitation. Five μg of nuclear and one-fifth of the immunoprecipitation input were used for western blotting. (C) Western blotting analysis of co-immunoprecipitated samples. 300 μg of nuclear lysate was mixed with FLAG M2 magnetic beads for immunoprecipitation. (D) Western blotting analysis of coimmunoprecipitated samples. 500 μg of nuclear lysate was combined with FLI1 antibody and protein A/G magnetic beads for immunoprecipitation. Five μg of nuclear lysate and one-fifth of the immunoprecipitation input were used for western blotting.

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The NR0B1 gene harbors EWS-FLI1-bound GGAA microsatellites within its promoter region [35]. We cloned the NR0B1 promoter region upstream of Nanoluc and measured NR0B1 promoter activity in siAHDC1-treated cells (Fig 3B). AHDC1 knockdown showed downregulation of NR0B1 promoter activity in A673 cells. In addition, the ChIP assay using cells expressing doxycycline-induced 3xEGFP, 3xFLAG-EWS-FLI1, or 3xFLAG-AHDC1 showed that EWS-FLI1 and AHDC1 could bind the GGAA microsatellite on the NR0B1 promoter (S5A Fig). We also measured mRNA levels of target genes of EWS-FLI1 by RT-qPCR in siAHDC1-treated cells (Fig 3C). mRNA expression of PPP1R1A, GLI1, FoxM1, and NR0B1 genes highly expressed in Ewing's sarcoma cells was dependent on EWS-FLI1 [35][36][37][38]. These genes were downregulated in siAHDC1-treated cells but not NKX2-2 and EWS-FLI1. We speculated that AHDC1 might affect EWS-FLI1 protein stability. EWS-FLI1 protein turnover is proteasome-dependent and may also be lysosomal-dependent [13,39]. In AHDC1 knockdown cells, EWS-FLI1 protein amount reduced rapidly compared to the negative control after treatment of Cycloheximide, a protein synthesis inhibitor (S5B Fig). EWS-FLI1 and NKX2-2 protein amount increased by adding MG-132, a proteasome inhibitor, as previously reported (S5C Fig) [39,40]. In AHDC1 knockdown cells, decreased EWS-FLI1 and NKX2-2 protein amount was not fully suppressed by MG-132 treatment. Although we do not examine the lysosomal degradation of EWS-FLI1, EWS-FLI1 might partially degrade in lysosome by independently proteasomal machinery, or AHDC1 might also regulate ubiquitination proteins. Alternatively, AHDC1 might affect the post-transcriptional machinery of EWS-FLI1 and NKX2-2. We need to elucidate this hypothesis in the future.
To determine whether the EWS-FLI1 protein is involved in AHDC1 gene expression, we performed an EWS-FLI1 knockdown (Fig 3D). AHDC1 protein expression level was not altered in shEWSR1 or shFLI1-treated cells. These results suggest that AHDC1 is involved in EWS-FLI1-mediated transcriptional regulation, at least by binding to the GGAA microsatellite region on the NR0B1 promoter. However, how AHDC1 regulates EWS-FLI1 protein levels still needs to be examined.

AHDC1 knockdown attenuates cell growth in Ewing's cells
EWS-ETS fusion proteins are essential for the cell growth of Ewing's sarcoma. To test whether AHDC1 affects cell growth in Ewing's sarcoma cells, we transduced shAHDC1-expressing lentivirus in A673 cells (S6 Fig). EWS-FLI1, NR0B1, and NKX2-2 protein expression were reduced in shAHDC1-expressing cells as well as in siAHDC1-treated cells. After lentivirus transduction, cells were collected and spread again onto the 96-well microplate, resulting in the reduction of cell growth (Fig 4A). In addition, the spheroid culture of shAHDC1-expressing cells also showed reduced cell growth in a 3D-culture well (Fig 4B). Seki and NCR-EW2 cells were also treated with shAHDC1-expressing lentivirus, resulting in the reduction of cell growth (S7A Fig). AHDC1 knockdown was performed in HEK293 or hTERT RPE-1 cells as non-Ewing's cell types (S7B Fig). NR0B1 was weakly expressed in both cell lines but did not alter after shAHDC1 transduction (S7B Fig). HEK293 and hTERT RPE-1 cells did not show reduced cell growth after shAHDC1 transduction (S7C Fig). In addition, SAOS-2 and U-2 OS osteosarcoma cell lines did not alter any growth defects after shAHDC1 knockdown, suggesting that AHDC1 affects cell growth in Ewing's sarcoma cells ( S8 Fig). Next, we assessed cell cycle progression and apoptotic activity after AHDC1 knockdown. siAHDC1-treated cells presented an increased p27 protein level (Fig 4C). In addition, shAHD-C1-expressing cells showed a high caspase activity level (Fig 4D). EWS-FLI1 knockdown shows increased cell adhesion, migration, and invasion in Ewing sarcoma cells [41][42][43]. AHDC1 knockdown and EWS-FLI1 knockdown up-regulated Snail and α-smooth muscle

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actin (α-SMA), both of which are the epithelial-mesenchymal transition marker (Fig 4E and  S9A Fig), but AHDC1 knockdown did not affect cell migration (S9B and S9C Fig). These results suggest that AHDC1 affects cell cycle progression and suppression of apoptosis in Ewing's sarcoma cells.

AHDC1 knockdown reduces BRD4 and BRG1 protein expression
In our proximal proteins screening of EWS-ETS fusion proteins, we also identified BRD4 and BRG1 (S2 Fig and S3 Table) [44]. BRD4 has been shown to interact with EWS-FLI1, and BRD4 inhibition by BET inhibitors results in reduced cell growth in Ewing's sarcoma cells [9,10,45,46]. EWS-FLI1 recruited BRG1/BRM-associated factor (BAF) complexes containing BRG1 to the GGAA microsatellite region [5]. We tested whether BRD4 and BRG1 protein expression levels are affected by AHDC1 knockdown (Fig 5A). AHDC1 knockdown showed reduced BRD4 and BRG1 protein expression levels. Fluorescent protein-tagged AHDC1 localized in the nuclei in Hela cells [47]. We expressed FLAG-tagged AHDC1 by using the piggyBac system under the control of the Tet-on system in A673 cells and stained with BRD4 and BRG1 (Fig 5B). AHDC1 was partially co-localized with endogenous BRG4 and BRG1 at the z-stack visualization. AHDC1 may contribute to stabilizing BRG1 and BRD4 proteins by co-localization. We need to clarify the relationship between AHDC1 and chromatin regulators in the future.

Discussion
Proximal protein identification using new tools such as APEX2, BioID, or their derivatives has been a promising tool for biochemical approaches in vitro or in vivo [11]. In this study, we isolated AHDC1 as a proximal protein of the EWS-ETS proteins using the screening of the BioID system. AHDC1 was necessary to grow Ewing's sarcoma cells but not non-Ewing's sarcoma cells such as HEK293 or hTERT RPE-1 cells. In addition, AHDC1 affected gene expression of EWS-FLI1 target genes. Thus, AHDC1 may be one of the regulators for oncogenic function in Ewing's sarcoma cells.
In BioID analysis using the three EWS-ETS fusion proteins, we identified 193 proteins ( Fig  1C), while the 366 proteins were previously identified in 293 cells [10]. Twenty-one proteins overlapped in our list. We thought of three possible reasons for this difference: first, differences in purification methods. We purified the biotinylated proteins with streptavidin sepharose, digested, and collected them with Trypsin/Lys-C on beads. In the previous paper, the biotinylated proteins were purified with streptavidin agarose and separated by SDS-PAGE, followed by in-gel digestion with Trypsin [10]. In-gel digestion may have reduced the number of identified protein amounts. Second, there are cellular differences. We used an Ewing's sarcoma cell line for BioID analysis, whereas the previous study used 293 cells [10]. These differences may be due to differences in gene expression patterns. Third, differences in negative controls. We used BioID and BioID-tagged Luc2 as negative controls. We searched protein amounts from precursor ions in each BioID sample and compared the amount of BirA(R118G) in each sample for normalization (Materials and methods). In previous data, negative controls were subtracted from the CRAPome database [44]. This difference may result in a difference in proteins identified by each BioID method. Our raw data of the BioID screening contained much contamination that non-specifically binds to streptavidin Sepharose beads because 3879 proteins were identified, but 193 proteins were only found as proximity proteins of EWS-ETS fusion proteins. Hence, the purification method needs to be improved, including the type of beads and washing method.

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We also checked BioID-tagged fusion protein expression. Doxycycline-induced BioIDtagged EWS-ETS proteins were highly expressed compared to endogenous EWS-FLI1 (S1A Fig). Although several proteins could be reproduced in the band after purification of streptavidin Sepharose without ARNT, RBM26, and IRX4 (S3 Fig), we do not subtract the truncated EWS or ETS fusion proteins as the negative control. We guess that our list might include proximal proteins of intact EWSR1, FLI1, ERG, and ETV4. In addition, excessive induction may lead to nonspecific biotinylation, and nonspecific biotinylated proteins may be included in the list of proteins we have identified.
The Xia-Gibbs syndrome has been identified as a de novo heterozygous truncating mutation of AHDC1 [14]. More than 100 cases of mutations related to the diagnosis of the Xia-Gibbs syndrome have been reported [15]. Not only heterozygous mutations of AHDC1 but also micro-duplication of the genome containing the AHDC1-coding region showed similar symptoms [52]. Thus, deregulation of AHDC1 gene expression affects the developmental process. AHDC1 has an AT-hook DNA binding motif, a PDZ motif, and other conserved domains within the coding sequence [47]. Feng et al. showed that AHDC1 expression was highly expressed in cervical cancer cells compared with immortalized cervical epithelium, and its expression was regulated by a long noncoding RNA, LINC01133 [53]. However, the molecular mechanisms for AHDC1 in cancer cells are still unclear.
EWSR1 is an RNA-binding protein comprising FET family proteins (FUS, TAF15, and EWSR1). EWSR1 is also one of the paraspeckle components that is a subcellular body in the nucleus and co-localized with SFPQ1, NONO, and PSPC1 [54,55]. AHDC1 was also isolated as one of the paraspeckle components co-localized with EWSR1 [54]. Khayat et al. showed that wild-type AHDC1 localized in the nucleus, and Xia-Gibbs patients with a mutation of AHDC1 have disrupted wild-type AHDC1 localization in HeLa cells [47]. Our proximal proteins screening of EWS-ETS fusion proteins did not isolate SFPQ, NONO, or PSPC1. However, CPSF5 (NUDT21), CPSF6, and CPSF7 were isolated as paraspeckle components and are the components of the cleavage factor Im (CFIm) complex that brings about cleavage of 3'UTR of mRNA for polyadenylation were isolated as proximal proteins of EWS-ETS fusion proteins (S2 Fig and S3 Table) [54]. These results suggest that some paraspeckle components may interact with transcriptional complexes with EWS-ETS fusion proteins.
FET family proteins comprising FUS, EWSR1, and TAF15 are not only involved in neurodegenerative disease but also act as oncoproteins in sarcoma or leukemia by chromosomal translocation. The N-terminal region of FET family proteins comprising SYGQ-rich regions interacts with the SWI/SNF chromatin remodeling complex containing BRG1 [5,28]. In our screening, the chromatin remodeling complex containing BRG1, ARID1A, SMARCC1, SMARCD1, SMARCE1, SMARCB1, and SMARCAL1 were isolated as proximal proteins of EWS-ETS fusion proteins ( S2 Fig and S3 Table). EWS-FLI1 recruits BRG1 to open the chromatin structure at the GGAA microsatellite region [5]. In our observations, AHDC1 contributed to maintaining the BRG1 protein expression level (Fig 5A and 5B). We postulate that the SWI/SNF chromatin remodeling complex may regulate EWS-FLI1 transcriptional activity with AHDC1. AHDC1 did not regulate the gene expression of EWS-FLI1 at the transcriptional level, while EWS-FLI1 protein was downregulated (Fig 3A and 3C). Our results also showed that a proteasomal inhibitor did not fully suppress the EWS-FLI1 protein level in AHDC1 knockdown (S5C Fig). AHDC1 might affect the lysosomal pathway for EWS-FLI1 protein level or might affect EWS-FLI1 protein level at post-transcriptional or protein synthesis level. We still need to continue this hypothesis. AHDC1 might stabilize BRD4 and BRG1 (Fig 5A). In addition, FLAG-tagged AHDC1 expression partially co-localized with BRD4 and BRG1 in Ewing's sarcoma cells (Fig 5B). We hypothesize that AHDC1 might be one of the accessory proteins needed to stabilize BRD4, BRG1, and probably EWS-FLI1 in Ewing's sarcoma cells.
Recently, Gibbin, a protein that encodes the AHDC1 gene, mediates connections between enhancers and promoters at the specific gene locus during development [50]. Proximity labeling of Gibbin showed that Gibbin mainly interacts with zinc-finger transcription factors and DNA methylation regulators. Gibbin loss caused hypermethylation and decreased CTCF deposition in BMP4/Retinoic acid-treated Human embryonic stem cells. ARID1A, FUBP1, and ZNF462 in Gibbin interactome were included in our EWS-ETS fusion proteins list. Although the protein size of the Gibbin and AHDC1 protein that we analyzed are different, we guess that AHDC1 might be one of the hubs between enhancers and promoters in the Ewing sarcoma cells and partially affect gene expression of the EWS-ETS fusion proteins.
Finally, we only performed experiments on AHDC1 using Ewing's sarcoma cell line, and we need to progress a cell line-derived xenograft model or patient-derived xenograft model using mice. In addition, we need to analyze the difference between AHDC1 low state and EWS-FLI1 low state because AHDC1 knockdown did not fully complete transcriptional activity by EWS-FLI1 knockdown in the future. promoter that harbors a GGAA microsatellite region was performed using KOD one polymerase with NR0B1-ChIP-f and NRoB1-ChIP-r primers at 35 cycles. The ChIP assay was performed in three independent replicas. (B) Cells after treatment of siAHDC1 RNA were performed for 2 d, treated with 20 μg/ml Cycloheximide for 8 h, and lysed by 1xSDS sample buffer. Each protein was detected by its respective antibody. (C) Cells after treatment of siAHDC1 RNA were performed for 2 d, treated with 10 μM MG-132 for 8 h, and lysed by 1xSDS sample buffer. Each protein was detected by its respective antibody.